Stem cell stimulating compositions and methods of treating melasma

ABSTRACT

Compositions and methods for topical treatment of the hyperpigmented lesions of melasma are presented. Such compositions include defensins in concentrations that are below those that exhibit antimicrobial activity, and can be in the form of a topically applied gel, lotion, wash, shampoo, cream, or mask. Various formulations for such compositions, which can include various pharmaceutically acceptable stabilizers, emollients, and fragrances, are provided.

FIELD OF THE INVENTION

The field of the invention is topical cosmetic formulations useful inthe treatment of melasma.

BACKGROUND

The following background discussion includes information that may beuseful in understanding the present invention. It is not an admissionthat any of the information provided herein is prior art or relevant tothe presently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Melasma is characterized by the development of well demarcated brown orgray-brown patches of hyperpigmentation, typically on the skin of theforehead, cheeks, and upper lip (although such hyperpigmented patchescan appear elsewhere on the body). Development of these dark patches ismore common in women, and is often associated with pregnancy, the use ofhormonal contraceptives, and hormone replacement therapy. Thedevelopment of melasma also appears to be associated with sun exposure,being most common in women with light brown skin coloration living insunny climates.

Although melasma is harmless, the appearance of these dark patches isconsidered to be cosmetically unappealing and can be a source ofdistress. While the appearance of melasma can be reduced usingcamouflage makeup, such an approach requires considerable skill andeffort, and may need to be repeated throughout the day. In manyinstances melasma will resolve spontaneously on discontinuation ofhormonal treatments, avoiding sun exposure, and application of sunblock. Currently, melasma is generally treated using topically applieddepigmenting agents (such as hydroquinone), chemical agents that alterthe activity of melanocytes, chemical or mechanical “peels” that removethe affected skin surface, and laser treatment. Unfortunately some ofthese treatments cannot be utilized during pregnancy. In additionreoccurrence is common, particularly following sun exposure.

Since their initial discovery in the 1960's, there has been muchresearch surrounding the role of defensins. Defensins are smallcysteine-rich proteins, usually only 14-85 amino acids long. Defensinscan be found in invertebrates, vertebrates, and plants, and have beenshown to be active against many bacteria, fungi, and viruses. In fact,much of the body of research has focused on the antimicrobial propertiesof defensins. However, in recent years, some research has explored otherroles defensins may play in human skin, such as in wound healing or hairgrowth.

PCT Patent Application WO 2014/004339 A2 by Applicant ELC Management LLCteaches the use of a resveratrol-containing cosmetic composition tostimulate endogenous production of cellular beta defensins in skincells. In order to illustrate this effect, the inventors tested thecomposition on Normal Human Epidermal Keratinocytes (“NHEK”) in vitroand measured the presence of beta defensin in NHEK. The resultingstimulated quantities of defensin were very small (approximately 0.001ng/ml). The inventors theorized that the stimulation of beta defensinsin keratinocytes by resveratrol-containing compositions would beeffective for treating acne, inhibiting microbial growth on the skin,and improving skin barrier repair. Because of the low stimulatedquantities of beta defensin, and because the compositions do notactually contain defensins, it is questionable that these compositionswould produce cosmetically meaningful results. These and all otherextrinsic materials discussed herein are incorporated by reference intheir entirety. Where a definition or use of a term in an incorporatedreference is inconsistent or contrary to the definition of that termprovided herein, the definition of that term provided herein applies andthe definition of that term in the reference does not apply.

On the other hand, defensins have been shown to play a significant rolein wound healing. In the journal article titled “Stimulation of thefollicular bulge LGR5+ and LGR6+ stem cells with the gut-derived humanalpha defensin 5 results in decreased bacterial presence, enhanced woundhealing, and hair growth from tissues devoid of adnexal structures”(Plast. Reconstr. Surg. 2013; 132(5):1159-71), Lough et al. report ahuman alpha defensin 5-containing formulation that was shown underexperimental conditions to recruit LGR5+ and LGR6+ stem cells tothird-degree burns in mice, which accelerated healing of the wound.However, these findings leave many questions. For example, in theexperimental conditions described by Lough et al., it is unclear ifLGR5+ and LGR6+ were activated by pro-inflammatory conditions and otherfactors already present in the wound or due to the topical applicationof defensins. Furthermore, the wound healing formulation used by Loughet al. contained concentrations of defensins at antimicrobially activeconcentrations (e.g. about 105 ng/ml). However, this high concentrationof defensin may make any resulting cosmetic composition more allergenicand more costly.

Therefore, even though defensin containing and defensin stimulatingcompositions are known, there is a need for a cosmetic topicalformulation that are effective in reducing or eliminating the occurrenceand/or appearance of melasma.

SUMMARY OF THE INVENTION

The inventive subject matter is directed towards various topicalformulations, methods of manufacture of the topical formulation in whichthe topical formulation includes antimicrobially effective orsub-antimicrobially effective concentrations of at least one defensin,and methods of applying the topical formulation to the healthy skin ofusers to reduce or eliminate the appearance of melasma (for example,epidermal melasma).

In one aspect of the invention, a topical cosmetic formulation includesa defensin in a cosmetically acceptable carrier. Preferred topicalcosmetic formulations are ready-to-use and contain the defensin at asub-antimicrobially effective concentration, wherein the concentrationis ineffective to inhibit growth of a microbial pathogen in atherapeutically effective manner but is effective in reducing oreliminating the appearance of melasma. Still further preferred topicalformulations may further comprise a blend of two or more differentdefensins, wherein the combined concentration of defensins in theformulation is a sub-antimicrobially effective concentration.

The inventors further contemplate methods of using defensins atantimicrobially effective or sub-antimicrobially effectiveconcentrations in topical formulations to recruit LGR6+ stem cells to aninterfollicular space in non-injured skin. It should be appreciated thatmethods of recruiting LGR6+ stem cells may include a step of providing atopical formulation containing a sub-antimicrobial concentration of atleast one defensin and a further step of applying the formulation tonon-injured skin to reduce or eliminate the appearance of melasma and/ora treated melasma lesion.

It is preferred that the inventive compositions, methods, and usesemploy at least one of alpha-defensin 1, alpha-defensin 5,alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2,neutrophil defensin 3, neutrophil defensin 4, theta-defensin,beta-defensin 1, beta-defensin 2, beta-defensin 3, and beta-defensin 4.In especially preferred topical compositions and methods, alpha-defensin5 and beta-defensin 3 are employed. It should be appreciated that thedefensin can comprise a synthetic defensin, a human defensin,recombinant defensin, a primate defensin, a murine defensin, a caprinedefensin, a bovine defensin, an ovine defensin, an equine defensin, alapine defensin, a porcine defensin, a canine defensin, and/or a felinedefensin.

With respect to the sub-antimicrobially effective concentration of thefirst defensin in the ready-to-use topical cosmetic formulation,contemplated concentrations may be between 0.01 and 100 ng/ml, orbetween 1 and 30 ng/ml, including the end points of each range.Additionally, especially preferred embodiments of the inventive subjectmatter employ defensin concentrations of about 22 ng/ml and about 4.4ng/ml in ready-to-use formulations.

The inventors unexpectedly found that, even at these low concentrations,defensins can be effective to substantially reduce the appearance ofmelasma and/or a treated melasma lesion. Without wishing to be bound byany particular theory, the effectiveness of defensins at thesesub-antimicrobially effective concentrations may be due to theactivation and/or recruitment of LGR6+ stem cells. Typically, defensinsused in preferred embodiments of the inventive subject matter have apurity greater than 95% as shown by HPLC, and the sequence and properdisulfide bond formation of the defensins can be confirmed by tandemMS/MS.

Depending on the nature of the topical formulation, it should berecognized that defensins may be encapsulated in liposomes or othernanoparticles. In preferred formulations, defensins may also beassociated with a carrier, in particular a protein carrier such asalbumin (e.g., human serum albumin, bovine serum albumin, egg albumin,and recombinant albumin produced by rice, other plants, bacteria oryeast), also encapsulated in liposomes where desirable.

The inventors further contemplate that the topical cosmetic formulationsmay also include supplements to provide nutrition and support for LGR6+stem cells. Typical supplements include human serum albumin, bovineserum albumin, egg albumin (i.e. ovalbumin), recombinant albuminproduced by rice, other plants, bacteria or yeast, plant hydrolysate,beta-cyclodextrin, glutamine, phospholipids, fibronectin, hyaluronate,hyaluronic acid, plant hydrolysate, L-alanyl-L-glutamine, gelatin,recombinant gelatin, Epidermal Growth Factor (EGF), vitamin E,Tocopheryl Nicotinate, ubiquinone, coenzyme Q10, and/or an antioxidant.Suitable cosmetic formulations can also incorporate an ultravioletand/or visible light blocking or screening agent.

The inventors have appreciated that the topical cosmetic formulations ofthe inventive subject matter can be included in kits with exfoliatingmasks. Especially preferred kits include a mask, a cream treatmentformulation, and a serum treatment formulation.

Further aspects of the inventive subject matter provide methods oftreating scars, sunburn, bruises, and other skin disorders in which theepidermal layers of the skin are largely intact. Exemplary methodsinclude the steps of providing a topical formulation having at least onedefensin at a sub-antimicrobially effective concentration and applyingthe formulation to non-injured skin under a protocol effective tosubstantially reduce the appearance of melasma an/or a treated melasmalesion.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show side-by-side photographs that show reduction inwrinkles. FIG. 1A shows the appearance of the skin prior to treatment.FIG. 1B shows the appearance of the skin following treatment.

FIGS. 2A and 2B show side-by-side photographs that show a reduction inthe appearance of brown spots. FIG. 2A shows the appearance of the skinprior to treatment. FIG. 2B shows the appearance of the skin followingtreatment.

DETAILED DESCRIPTION

The inventors unexpectedly discovered that antimicrobal andsub-antimicrobial concentrations of defensins can achieve numerousdesirable effects on non-injured skin when applied in ready-to-usetopical formulations. Among other things, such topical formulationsprovide a significant reduction or elimination of the occurrence and/orappearance of the hyperpigmented lesions characteristic of melasma, forexample epidermal melasma.

In one preferred embodiment, a ready-to-use topical cosmetic formulationcomprises at least one defensin present in a topical cosmeticformulation at a sub-antimicrobially effective concentration. Of courseit should be appreciated that topical formulations may contain onedefensin, a combination of two defensins, or a combination of three ormore defensins. The defensins used may be of the same or different typesand subtypes. For example suitable defensins may include one or more ofalpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophildefensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophildefensin 4, theta-defensin, beta-defensin 1, beta-defensin 2,beta-defensin 3, and beta-defensin 4. Especially preferred topicalformulations contain alpha-defensin 5 and beta-defensin 3. When two moredefensins are used in combination, each defensin may be present in equalquantities by mass or at mass ratios specified to achieve a desiredresult, such as 1:1.5, 1:2, 1:4, 1:5, etc. Notably, it should beappreciated that the total concentrations of defensins used incontemplated ready-to-use formulations are ineffective at inhibitingsubstantial proliferation of microbes in established skin infections ina therapeutically effective manner. Alternatively, in other embodimentsof the inventive concept the concentration of defensin or defensinscomprising the formulation can be sufficient to have an anti-microbialeffect.

As used herein, the term “ready-to-use” indicates that thedefensin-containing topical formulation is in a form that is presentedfor sale and application. It is contemplated that ready-to-useformulations can comprise a fully combined solution, cream, gel, serum,lotion, etc. Alternatively, the defensin can be packaged in a separatecontainer (e.g., in a vial that pumps a defensin solution with a creamthat the user blends before applying to unbroken skin) and combined withanother topical formulation at the time of use/application.

As also used herein, the phrase “sub-antimicrobially effectiveconcentration” means concentration(s) of defensins which arecharacterized by an inability to inhibit the proliferation of microbesin an established infection. Typically, ready-to-use topicalformulations do not include concentrations greater than 1 μg/ml. Inpreferred embodiments, the concentration of defensins lies between about0.01 and about 100 ng/ml, and even more typically between about 1 andabout 30 ng/ml, wherein contemplated concentrations include the endpoints of each range. In even more preferred embodiments, the topicalcosmetic formulations have defensin concentrations of about 22 ng/ml andabout 4.4 ng/ml. As used herein, when the term “about” is used inconjunction with a numeral, “about” means a range of plus or minus tenpercent of the numerical value given, including end points. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein.

General topical formulations can include any and all formulationssuitable for cosmetic topical use, especially on non-injured skin. Asused herein, the term “non-injured” skin refers to skin in which dermisand hypodermis are substantially intact. Therefore, viewed from adifferent perspective, non-injured skin will appear intact to theunaided eye, with no breach sufficiently large or deep to result inbleeding. Thus, non-injured (or “healthy”) skin includes aged skin andskin with first degree sunburn, environmental exposure, bruising, orpartially ablated stratum corneum. Non-injured (or healthy) skin alsoexcludes skin displaying persistent infection with pathogens that resultin visible symptoms and signs of infection.

With respect to the source of defensins, the inventors contemplate thatdefensins from both natural and synthetic sources may be suitable forincorporation into topical formulations. For example, defensins may beobtained from plants (e.g., Arabidopsis, pea, tobacco, spruce, and/orrecombinant plants), mammals or other animals, recombinant organisms(such as yeast or bacteria), and/or products of laboratory peptidesynthesis (for example, through the use of a Merrifield resin or othersolid-phase synthesis). Exemplary defensins derived from natural sourcesmay include human defensins, simian defensins, murine defensins, bovinedefensins, ovine defensins, caprine defensins, equine defensins, lapinedefensins, porcine defensins, canine defensins, and/or feline defensins.

Due to their relatively low quantities in a living organism and lowmolecular weight, it is generally preferred, however, that the defensinsare synthetic defensins. Synthetic defensins include defensins producedby chemical synthesis (e.g., solid phase synthesis) or by recombinanttechnologies (e.g., produced by recombinant bacteria, yeast, tissuecultures, plants or animals). The inventors further contemplate thatdefensin analogues such as hapivirins and diprovirins may be used insome embodiments of the inventive subject matter. Still further, theinventors further contemplate that the defensins can also be modified toincrease their activity and specificity for cosmetic improvements to theappearance of skin. For example, defensins may be unfolded and refoldedunder controlled conditions to ascertain proper disulfide bond formation(which can be monitored by MS analysis and/or CD spectroscopy).Alternatively, chemical modifications (e.g., using non-natural aminoacids and/or PEGylation to increase half-life time, or derivatizedproteinogenic amino acids to increase lipophilicity) are contemplated totailor the defensins to a particular need.

Regardless of the source of the defensins, it should be appreciated thatspecific activity of defensins is dependent on various factors,including isomeric form and tertiary structure of the final protein.Thus, and especially where the defensin is synthetic, orthogonalprotecting groups can be used to protect selected cysteine residues,which can then be individually deprotected and bonded with the matchingtarget cysteine residue, leading to coordinated non-random disulfidebond formation. Use of such protecting groups in the synthetic strategycan give rise to defensins with a specific activity that is comparableto the specific activity of the native defensin. Any suitablecharacterization and quality control measures may be employed.Typically, the specific activity of defensins incorporated into theinventive topical formulations is measured by purity as determined byHPLC. In exemplary embodiments, the defensin is between about 80% andabout 100% pure, more typically the defensin is at least about 90% pure,or at least about 95% pure, or at least about 99% pure, or at leastabout 99.9% pure. Additionally, proper amino acid sequence and disulfidebond formation can be confirmed by tandem MS/MS, for example.

With respect to suitable concentration of defensins in cosmeticformulations presented herein, it is contemplated that allconcentrations are deemed appropriate so long as such concentrations arecosmetically effective in reducing the appearance and/or occurrence ofhyperpigmented regions characteristic of melasma on healthy skin.Consequently, the total concentration of defensins (single type orcombination of distinct defensins) in the final cosmetic formulation asapplied to the skin can be between about 0.01 ng/ml and about 100 ng/ml,or between about 0.1 ng/ml and about 100 ng/ml, or between about 1 ng/mland about 100 ng/ml, or between about 2 ng/ml and about 80 ng/ml, orbetween about 4 ng/ml and about 60 ng/ml, or between about 1 ng/ml andabout 30 ng/ml. Thus, preferred compositions include defensins at aconcentration of at least about 0.01 ng/ml, at least about 0.1 ng/ml, atleast about 1 ng/ml, or at least about 4 ng/ml, but no more than about200 ng/ml, no more than about 100 ng/ml, no more than about 75 ng/ml, orno more than about 50 ng/ml.

In some embodiments the defensin can be associated with a cosmeticallyacceptable protein to increase stability and/or deliverycharacteristics. In this context, it should be appreciated that such anassociation is preferably non-covalent (e.g., electrostatic, ionic,hydrophobic, etc.), however, covalent attachment to a side group of theprotein is not excluded. Exemplary cosmetically acceptable proteinsinclude lactoferrin, transferrin, and albumin (e.g., human serumalbumin, bovine serum albumin, ovalbumin, and/or recombinant albumin).The defensins and protein carriers can be in various ratios, includingequimolar, sub-, and supramolar ratios. Additionally, combinations oftwo or more protein carriers can be used. For example, in a formulationin which two defensins are used, one defensin may be associated with onecarrier, and the other defensin may be associated with a differentcarrier. Therefore, any combination of defensins and carriers iscontemplated.

In still further contemplated aspects, the defensins (and carrierproteins) can be encapsulated in cosmetically acceptable formulations,and especially formulations using a lipid membrane. For example,liposomes, microcapsules, nanocapsules, microparticles, nanoparticles,microparticle delivery systems, are especially contemplated. Adescription of some cosmetically acceptable cosmetic delivery systemscan be found in Maherani et al, “Liposomes: A Review of ManufacturingTechniques and Targeting Strategy,” Current Nanoscience; 7:436-452(2011). A preferred method of liposome manufacturing is a shear method.Preferred cosmetic delivery systems resemble naturally occurringmembranes, are flexible, and can penetrate interstitial spaces betweencells. It is further contemplated that cosmetic delivery systems mayhave monolayer, bilayer (e.g. unilammellar vesicle or ULV), or multilayer structures (e.g. multilamellar vesicle or MLV). Additionally,multilayer liposomes, microcapsules, microsomes, and nanocapsules canhave nested structures (e.g. multivesicular vesicle or MVV). Cosmeticdelivery systems used in the topical formulations can range in size fromabout 500 nm to about 10 μm. In the preparation of cosmetic deliverysystems, all cosmetically acceptable lipid compositions arecontemplated, especially pharmaceutically acceptable lipids. In mostinstances preferred cosmetic delivery systems comprise amphipathic oramphiphilic molecules such as phospholipids or combinations ofphospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine,phosphatidic acid, phosphatidylserine, and phosphoinositides).Additionally, in some instances contemplated cosmetic delivery systemscan contain additive(s) such as sterols, polyethylene glycol,cholesterol, dicethylphosphate, stearyl amine, etc. With respect to theamount of delivery systems incorporated in each ready-to-useformulation, the cosmetic delivery system content will typically beadjusted to achieve a sub-antimicrobial concentration of defensinswithin a preferred range. Unilamellar vesicles/liposomes can be producedusing high shear techniques. These vesicles have a greater ZetaPotential than the typical liposome, which allows for smaller, moreuniform particle size with increased stability. Zeta Potential is anindicator of the electronic charge on the surface of any macroscopicmaterial that is in contact with a liquid. This can be used to predictand control the stability of suspensions; the higher the Zeta Potential,the greater the stability of the molecule because the charged particlesare able to repel and overcome their innate affinity to assemble.

It should be noted that defensins, protein carriers, liposomes, or othermembranaceous structures have a molecular weight that exceedstransmembrane delivery, and even delivery across the stratum corneum.Nevertheless, as is discussed in more detail, the defensins have aprofound effect on stem cell activity in dermal and hypodermal layers.While not wishing to be bound by any theory or hypothesis, the inventorscontemplate that the liposomal formulations have the ability totransport the defensins via an interstitial route and/or to invade thehair follicle to a depth and concentration sufficient to activate LGR6+cells. Viewed from another perspective, the use of cosmetic deliverysystems is thought to aid in the delivery of defensins as one would notexpect defensins per se to penetrate the stratum corneum of the skin(acting as a barrier to molecules with molecular weights greater than500 Da). Moreover, when associated with albumin (65-70 kDa) and/orliposomes, it becomes at least conceptually even more difficult for thedefensin composition to penetrate unbroken skin. Thus, the inventorshypothesize that the mechanism for delivering defensins is different inunbroken skin when compared with broken or injured skin.

In some embodiments of the inventive concept a formulation that includesone or more defensins can be delivered by an active mechanism. Suitableactive mechanisms can include application of laser energy, acousticpressure wave, ultrasound, microneedles, and/or injections to the skinarea undergoing treatment, in order to facilitate transport of thedefensins to the desired region of the skin and/or skin-associated (i.e.epidermis, dermis, basal layer, and/or hair follicle) area(s).

In another aspect, the inventors advantageously provide additionalingredients that nourish and support the recruited stem cells in healthyskin. For example, albumin (e.g., human serum albumin, bovine serumalbumin), egg albumin (ovalbumin), recombinant albumin, planthydrolysate, and β-cyclodextrin, glutamine, phospholipids (liposomes),fibronectin, hyaluronate, plant hydrolysate, L-alanyl-L-glutamine,gelatin, Vitamin E (tocopheryl nicotinate), ubiquinone (coenzyme Q10),gelatin, recombinant gelatin, hyaluronic acid, Epidermal Growth Factorcan provide nutrition and support to the stem cells.

In yet further contemplated aspects, cosmetic treatment of melasma onhealthy skin can be further be assisted by supplemental procedures.Especially contemplated procedures include chemical and/or mechanicalexfoliation. For example, chemical exfoliation may be performed usingone or more proteases (for example papain, Lactobacillus/Pumpkin FermentExtract, Lactobacillus/Punica Granatum Fruit Ferment Extract),alpha-hydroxy acids, etc. while mechanical exfoliation may be performedusing sugar crystals, cellulosic plant matter, tape stripping, frozenCO₂, polymeric beads, and/or silica granules.

Although not wishing to be bound by theory, the inventors contemplatethat the inventive subject matter (i.e., methods and use of antimacribically effective or sub-antimicrobially active concentrations of defensinsin topical cosmetic formulations) can act to recruit LGR6+ stem cells tothe interfollicular space in non-injured skin. Most typically, a userwill be instructed to apply the topical formulation to regions ofnon-injured skin that include or are likely to include hyperpigmentedareas characteristic of melasma under a protocol effective to reduce oreliminate the appearance of hyperpigmented areas of skin, and/oreffective to reduce or eliminate the occurrence of at suchhyperpigmented areas. For example, defensins can be applied at leastonce daily (or twice daily) for a period of at least one week, or twoweeks, three weeks, six weeks, or even longer. Beneficially, the totalquantity of applied formulations is such that the formulation isabsorbed into the skin. For example, topical formulations are typicallyapplied at about 0.1 to about 500 mg per cm², about 0.1 to about 500 mgper cm², about 0.5 to about 300 mg per cm², about 5 to about 500 mg percm², or about 100 to about 500 mg per cm².

Using topical compositions such as those presented in the examplesbelow, the inventors discovered that cosmetic formulations that includedthe defensins are effecting in reducing the appearance and/or preventingthe occurrence or reoccurrence of hyperpigmented lesions associated withmelasma.

In some embodiments of the inventive concept, topical application ofsuch defensin formulations on a suitable schedule is sufficient toreduce the area of a treated melasma lesion to less than 90%, 80%, 70%,60%, 50%, 40%, 30%, 20%, 10%, 5%, or less than 5% of the melasma lesionprior to treatment with the topical defensin formulation. In otherembodiments of the inventive concept, topical application of such adefensin formulation on a suitable schedule can reduce excessivepigmentation associated with a treated melasma lesion to less than 300%,200%, 100%, 50%, 25%, or 10% of pigmentation beyond that associated withnormal, unaffected skin adjacent to the treated melasma lesion.

In suitable treatment protocols the defensin formulation can be appliedfour times a day (e.g. every 6 hours), three times a day (e.g. every 8hours), twice a day (e.g. every 12 hours), once a day (e.g. in themorning, or prior to ultraviolet exposure), every other day, every threedays, once a week, or at a frequency determined through practical use.In some embodiments the topical defensin formulation can be applied morefrequently at the beginning of treatment. In other embodiments thetopical defensin formulation can be applied more frequently as treatmentprogresses. In still other embodiments treatment can continue on a firsttreatment schedule until the appearance of the melasma lesion iscompletely resolved, acceptable, and/or is stabilized and improved, andthen switched to a second treatment schedule that maintains the treatedappearance.

Treatment with the taught topical defensin formulations can be carriedout for any period of time suitable to eliminate or reduce theappearance of a melasma lesion. Suitable treatment periods can be atleast a week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4months, 5 months, 6 months, or longer. In some embodiments a topicaldefensin formulation can be applied on a long term (i.e. greater thanone year) or for an open ended time period on a maintenance schedulesuitable to provide and maintain the desired reduction or elimination inthe appearance of a treated melasma lesion.

Examples

Suitable formulations are described in International Patent ApplicationNo. PCT/US15/36049, which is incorporated herein by reference. Whilesuitable cosmetic formulations may be prepared using any number ofingredients and formulations known in the art, preferred topicalformulations include those that are ready-to-use and can be applied by auser. Therefore, with respect to cosmetically acceptable carriers, allcosmetically acceptable carriers are contemplated and include creams,oil-in-water emulsions, water-in-oil emulsions, foams, mousses,ointments, lotions, suspensions, serum, and gels. In some embodimentsthe cosmetic formulation can include a sunscreen, such as an ultravioletand/or visible light blocking ingredient (for example, zinc oxide),which advantageously reduces the impact of a suspected causative factorin the development of melasma.

Examples of suitable topical cosmetic cream formulations can include oneor more of the following ingredients: water, Carthamus tinctorius(safflower) oleosomes, Butyrospermum parkii (Shea) Butter, Macadamiaintegrifolia seed oil, niacinamide, yeast extract, ammoniumacryloyldimethyltaurate/VP copolymer, Helianthus annuus (sunflower) seedoil, phospholipids, alpha-defensin 5, beta-defensin 3, hyaluronic acid,Ophiopogon japonicus root extract, hydrolyzed Candida saitoana extract,sea whip extract, Lycium chinense fruit extract, Vaccinium angustifoliumfruit extract, Vaccinium marcrocarpon (cranberry) fruit extraubiquinone,L-alanyl-L-glutamine, Leuconostoc/Radish root ferment filtrate, gelatin,SH oligopeptide-1, xanthan gum, phytic acid, polysorbate 20,caprylic/capric triglyceride, phenoxyethanol, caprylyl glycol,ethylhexylglycerin, hexylene glycol, potassium sorbate, sodium chloride,and natural and/or artificial fragrance.

Examples of a suitable topical cosmetic serum formulation can includeone or more of the following: water, cyclopentasiloxane, glycerin,niacinamide, Sinorhizobium meliloti ferment filtrate, dimethicone,polysorbate 20, dimethicone/vinyl dimethicone crosspolymer, lauryl PEG-9polydimethylsiloxyethyl dimethicone, ammonium acryloyldimethyltaurate/VPcopolymer, phospholipids, alpha-defensin 5, beta-defensin 3, palmitoyltripeptide-38, sodium hyaluronate, Arabidopsis thaliana extract, seawhip extract, ergothioneine, Helianthus annuus (sunflower) seed oil,Rosmarinus offinalis (rosemary) leaf extract, SH oligopeptide-1,tocopheryl acetate, ubiquinone, Leuconostoc/radish root fermentfiltrate, albumin, gelatin, L-alanyl-L-glutamine, caprylic/caprictriglyceride, cetyl hydroxyethylcellulose, lecithin, hydroxypropylcyclodextrin, phytic acid, phenoxyethanol, caprylyl glycol,ethylhexylglycerin, hexylene glycol, and/or sodium chloride.

Examples of a suitable mask formulation can include one or more of thefollowing: butylene glycol, PEG-8, tapioca starch, sucrose, titaniumdioxide, hydroxyethyl acrylate/sodium acryloyldimethyl tauratecopolymer, squalane, polysorbate 60, Carica papaya (papaya) fruit,papain, Aloe barbadensis leaf juice, Lactobacillus/pumpkin fermentextract, Lactobacillus/Punica granatum fruit germent extract, sea whipextract, Cananga odorata flower oil, Citrus aurantium dulcis (orange)peel oil, caprylic/capric triglyceride, lactic acid, phenoxyethanol,caprylyl glycol, ethylhexylglycerin, and/or hexylene glycol.

Liposome Formulations

Table 1 below shows an exemplary cosmetic delivery liposome formulationincluding two types of defensins. These liposomes are typicallyincorporated into a cosmetic formulation at a fraction of about 1.0 wt %to 10.0 wt % for most skin care applications. In some embodimentsdefensin concentration can be up to 10 mg/ml or higher.

TABLE 1 Exemplary Liposome Formulation Component Concentration Water(protease-free) to 100% Albumin 0.1-1.0 mg/ml L-alanyl-L-glutamine0.1-1.0 mg/ml Gelatin 2-200 μg/ml Matrix proteins 1-100 ng/ml Humanalpha-defensin 5 1-200 ng/ml Human beta-defensin 3 1-200 ng/ml Growthfactors (e.g., EGF, FGF-2/ 0.1-100 ng/ml with or without) Phospholipids2-20 wt % Antioxidants 0.3-3 wt %

In yet a further aspect of the inventive subject matter, topicalcosmetic formulations can be offered together as a kit. Preferred kitsinclude a mask formulation and a defensin-containing cream or serumtreatment formulation. Even more preferred kits include a maskformulation and both the defensin-containing cream and serum treatmentformulations.

In the manufacture of cosmetic defensin formulations, it is contemplatedthat concentrated defensin preparations can be added to cosmetic baseformulations such that the concentration of the defensin in theready-to-use product is at a targeted sub-antimicrobially effectiveconcentration. Depending on the desired formulation, defensins can beincorporated into concentrated preparations as solutions, associatedwith carrier proteins, and more typically as liposomal formulations.Such concentrated defensin preparations can be added to the cosmeticbase formulations in proportions as given below:

Exemplary Body Lotion (Oil-in-Water) Formulation:

Mixture a) PEG-7 hydrogenated castor oil 2.00% PEG-20 glyceryl laurate1.00% cocoglycerides 3.00% cetearyl alcohol 1.00% cetearyl isononanoate4.00% octyl stearate 4.00% phenoxyethanol, methylparaben, 0.30%ethylparaben, butylparaben, propylparaben, isobutylparaben Mixture b)water, distilled 73.40% phenoxyethanol, methylparaben, 0.30%ethylparaben, butylparaben, propylparaben, isobutylparaben, glycerin3.00% Mixture c) concentrated defensin preparation 5.00% Mixture d)acrylamides copolymer, mineral oil 3.00% C13-C14 isoparaffin,polysorbate 85

To prepare the body lotion formulation mixture a) is melted atapproximately 70° C. Mixture b) is heated to approximately 70° C. andthen added to mixture a) while stirring. Stirring is continued until thelotion has cooled down to approximately 30 C°. Mixtures c) and d) areadded to the cooled mixture while stirring, and the lotion ishomogenized. In some embodiments final defensin concentration can rangefrom 1 ng/ml to 10 mg/ml or higher.

Exemplary Gel-Lotion Formulation:

Mixture a) acrylamides copolymer, mineral oil, 5.00% C13-14 isoparaffin,polysorbate 85 myreth-3 myristate 4.00% Mixture b) water, distilled85.00% phenoxyethanol (and) methylparaben (and) 0.50% ethylparaben (and)butylparaben (and) propylparaben (and) isobutylparaben xanthan gum 0.50%Mixture c) concentrated defensin preparation 5.00%

To prepare the gel lotion formulation mixture a) is dissolved atapproximately 50° C. Mixture b) is dispersed at room temperature andadded to mixture a) while stirring. Then, mixture c) is added whilestirring.

Exemplary Oil-in-Water Cream Formulation:

Mixture a) cetearyl alcohol (and) ceteareth-20 8.00% cocoglycerides2.00% cetearyl alcohol 2.00% dicaprylyl ether 8.00% oleyl erucate 7.00%phenoxyethanol, methylparaben, 0.30% ethylparaben, butylparaben,propylparaben, isobutylparaben Mixture b) water, distilled 62.40%phenoxyethanol, methylparaben, 0.30% ethylparaben, butylparaben,propylparaben, isobutylparaben glycerin 5.00% Mixture c) concentrateddefensin preparation 5.00%

To prepare the formulation mixture a) is melted at approximately 70° C.Mixture b) is heated to approximately 70° C. and added to mixture a)while stirring. Stirring is continued until the cream has cooled toapproximately 30° C. Then, composition c) is added while stirring andthe cream is homogenized. In some embodiments final defensinconcentration can range from 1 ng/ml to 10 mg/ml or higher.

Exemplary Water-in-Oil Cream Formulation:

Mixture a) diisostearoyl polyglyceryl-3 dimer dilinoleate 3.00% beeswax0.60% castor oil, hydrated 0.40% paraffinum subliquidum 5.00%isohexadecane 10.00% PPG-15 stearyl ether 2.00% dimethicone 0.50%phenoxyethanol, methylparaben, 0.30% ethylparaben, butylparaben,propylparaben, isobutyparaben Mixture b) water, distilled 68.40%phenoxyethanol, methylparaben, 0.30% ethylparaben, butylparaben,propylparaben, isobutylparaben glycerin 3.00% MgSO₄*7H₂O 1.00% Mixturec) concentrated defensin preparation 5.00% Mixture d) silica dimethylsilylate 0.50%

To prepare the oil in water cream formulation mixture a) is heated toapproximately 80° C. Mixture b) is brought to 80° C. and then added tomixture a) while stirring. Stirring is continued until the cream hascooled down to approximately 30° C., then mixtures c) and d) are addedand the cream is homogenized. In some embodiments final defensinconcentration can range from 1 ng/ml to 10 mg/ml or higher.

Exemplary Shampoo Formulation:

Sodium polyoxyethylene lauryl ether sulfate 15.0% Alkyl polyglucoside4.0% N-ethanol-N-methyl dodecanoic acid amide 3.0% EDTA-Na₂ 0.3% Malicacid to adjust pH to 6.0 q.s. Preservative 0.5% Concentrated defensinpreparation 10.0% Purified water balance Total 100.0%

To prepare the shampoo formulation all ingredients are mixed togetherand the volume is brought to about 90 ml. The pH is then adjusted andthe volume is finally adjusted to 100 ml (all percentages are weight %).In some embodiments final defensin concentration can range from 1 ng/mlto 10 mg/ml or higher.

Exemplary Body Wash Formulation:

Sodium polyoxyethylene lauryl ether sulfate 16.0% Sodium polyoxyethylene5.0% N-ethanol-N-methyl palm kernel oil fatty acid 2.5% amide Glycerin3.0% Cationized cellulose 0.1% Ethylene glycol distearate 3.0% EDTA-Na₂0.3% Citric acid to adjust pH to 5.7 q.s. Preservative 0.5% Concentrateddefensin preparation 7.5% Purified water balance Total 100.0

To prepare the body wash formulation all ingredients are mixed togetherand the volume is brought to about 90 ml. The pH is then adjusted andthe volume is finally adjusted to 100 ml (all percentages are weight %).In some embodiments final defensin concentration can range from 1 ng/mlto 10 mg/ml or higher.

Exemplary Face Wash Formulation:

Sodium polyoxyethylene lauryl ether sulfate 20.0% N-ethanol-N-methyldodecanoic acid amide 4.8% Glycerin 3.0% Hydroxyethyl cellulose 0.3%Ethylene glycol distearate 1.5% EDTA-Na₂ 0.3% Citric acid to adjust pHto 6.0 q.s. Preservative 0.5% Concentrated defensin preparation 10.0%Purified water balance Total 100.0

To prepare the face wash formulation all ingredients are mixed togetherand the volume is brought to about 90 ml. The pH is then adjusted andthe volume is finally adjusted to 100 ml (all percentages are weight %).In some embodiments final defensin concentration can range from 1 ng/mlto 10 mg/ml or higher.

The inventors tested a preferred formulation of the inventive subjectmatter in two clinical studies, Clinical Study 1 and Clinical Study 2(double-blinded study). Both studies were performed under thesupervision of Dr. Gregory Keller, M.D, F.A.C.S at the Plastic SurgeryClinic in Santa Barbara, Calif. The design for both studies wassubstantially similar. Both clinical studies examined 10 subjects over aduration of 6 weeks. Each subject was given a cream, a serum, and amasque. Half of the subjects (Study 2) were given formulations of thecream, serum, and masques containing defensins. The other half ofsubjects were given placebo formulations of the cream, serum, and masquethat were identical in composition to the test group, except theformulations did not contain defensins.

The inventors measured individual skin health scores for eachparticipant before and after treatment using the QuantifiCare™ 3DLifeViz™ Imaging Clinical System and protocol developed by QuantifiCareInc. (www.quantificare.com). The faces of each participants were scannedusing the 3D LifeViz™ system and given a value for each the followingcategories: wrinkle depth, length, and width; pore depth; skin evenness;skin oiliness; skin brown spots; and skin red spots. The resultingvalues for each participant were then compared against a population withthe same gender, age, and skin type, using QuantifiCare's ReferencePopulation Database. The resulting skin heath score for each participantwas a percentile ranking of skin health when compared with a populationof people with the same age, gender, and skin type.

For example, Table 2 (below) shows the average age of each study groupwhen compared to a population with corresponding age, gender, and skintype. Age was calculated using the wrinkle parameter, which is acombination of depth, length, and width of wrinkles in the forehead andcheeks.

TABLE 2 Average Age Ranking For Clinical Study 2 Before and AfterTreatment Average Age Ranking Group Before Treatment After 6 WeeksPlacebo 77% 74% Test Group 61% 81%

FIGS. 1A and 1B show the before (FIG. 1A) and after (FIG. 1B) imagesacquired by a 3D LifeViz™ system for participant 3-PC (63 year oldfemale) of Clinical Study 1. Participant 3-PC was given the testformulation. Measurements by the 3D LifeViz™ system showed thatparticipant 3-PC saw a reducing in visible skin age estimation (based onthe skin evenness value calculated by the 3D LifeViz™ system) from 64years to 37 years. Photos have not been retouched.

FIGS. 2A and 2B show before (FIG. 2A) and after (FIG. 2B) imagesacquired by a 3D LifeViz™ system with the brown-spot filter forparticipant 8-IK (female) of Clinical Study 1. Participant 3-PC wasgiven the test formulation. Images show a reduction in brown spots onthe face as a result of treatment. Photos have not been retouched.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc. Moreover, as used in thedescription herein and throughout the claims that follow, the meaning of“a,” “an,” and “the” includes plural reference unless the contextclearly dictates otherwise. Also, as used in the description herein, themeaning of “in” includes “in” and “on” unless the context clearlydictates otherwise.

1.-87. (canceled)
 88. A topical cosmetic formulation, comprising: acosmetically acceptable carrier in combination with a first defensin;wherein the first defensin is present in the topical cosmeticformulation at a concentration effective to reduce appearance of amelasma lesion, and wherein the topical cosmetic formulation is aready-to-use topical cosmetic formulation.
 89. The topical cosmeticformulation of claim 88, wherein the first defensin is selected from thegroup consisting of alpha-defensin 1, alpha-defensin 5, alpha-defensin6, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3,neutrophil defensin 4, theta-defensin, beta-defensin 1, beta-defensin 2,beta-defensin 3, and beta-defensin
 4. 90. The topical cosmeticformulation of claim 88, wherein the first defensin is selected from thegroup consisting of a synthetic defensin, a human defensin, recombinantdefensin, a simian defensin, a murine defensin, a caprine defensin, abovine defensin, an ovine defensin, an equine defensin, a lapinedefensin, a porcine defensin, a canine defensin, and a feline defensin.91. The topical cosmetic formulation of claim 88, wherein theconcentration of the first defensin in the ready-to-use topical cosmeticformulation is between 0.01 and 100 ng/ml, inclusive.
 92. The topicalcosmetic formulation of claim 88, further comprising a second defensin,wherein the second defensin is different from the first defensin. 93.The topical cosmetic formulation of claim 88, further comprising asupplement for LGR6+ stem cells, wherein the supplement is selected fromthe group consisting of human serum albumin, recombinant albumin, bovineserum albumin, egg albumin, plant hydrolysate, beta-cyclodextrin,glutamine, phospholipids, fibronectin, hyaluronate, hyaluronic acid,epidermal growth factor, fibroblast growth factor, plant hydrolysate,L-alanyl-Lglutamine, gelatin, recombinant gelatin, vitamin E, TocopherylNicotinate, Coenzyme Q10, and ubiquinone.
 94. The topical cosmeticformulation of any one of claims 88 to 100, wherein melasma is epidermalmelasma.
 95. A method of recruiting LGR6+ stem cells to aninterfollicular space in non-injured skin, comprising: providing atopical formulation comprising a first defensin at a concentrationeffective to reduce appearance of a lesion associated with melasma; andinstructing a user to apply the topical formulation to non-injured skinunder a protocol effective to reduce appearance of a lesion associatedwith melasma.
 96. The method of claim 95, wherein the first defensin isselected from the group consisting of alpha-defensin 1, alpha-defensin5, alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2,neutrophil defensin 3, neutrophil defensin 4, theta-defensin,beta-defensin 1, beta-defensin 2, beta-defensin 3, and beta-defensin 4.97. The method of claim 95, wherein the concentration of the firstdefensin in the topical formulation is between 0.01 and 100 ng/ml,inclusive.
 98. The method of claim 95, wherein the topical formulationfurther comprises a second defensin, wherein the second defensin isdifferent from the first defensin.
 99. The method of claim 95, whereinthe topical formulation further comprising a supplement for LGR6+ stemcells, wherein the supplement is selected from the group consisting ofhuman serum albumin, bovine serum albumin, egg albumin, planthydrolysate, beta-cyclodextrin, glutamine, phospholipids, fibronectin,hyaluronate, plant hydrolysate, L-alanyl-L-glutamine, gelatin, vitaminE, recombinant albumin, hyaluronic acid, epidermal growth factor,fibroblast growth factor, recombinant gelatin, Tocopheryl Nicotinate,Coenzyme Q10, and ubiquinone.
 100. The method of claim 95, wherein theprotocol comprises delivery of the first defensin to skin and/orskin-associated tissues using a device selected from the groupconsisting of a laser, acoustic pressure wave device, ultrasound device,a microneedle, and a hypodermic needle.
 101. A method of reducingappearance a lesion associated with melasma in non-injured skincomprising: providing a topical formulation comprising a first defensinat a concentration effective to recruit LGR6+ stem cells to aninterfollicular space in non-injured skin; and applying the topicalformulation to the non-injured skin under a protocol effective to reduceappearance of a lesion associated with melasma.
 102. The method of claim101, wherein the first defensin is selected from the group consisting ofalpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophildefensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophildefensin 4, theta-defensin, beta-defensin 1, beta-defensin 2,beta-defensin 3, and beta-defensin
 4. 103. The method of claim 101,wherein the concentration of the first defensin in the topicalformulation is between 0.01 and 100 ng/ml, inclusive.
 104. The method ofclaim 101, wherein the topical formulation further comprises a seconddefensin, wherein the second defensin is different from the firstdefensin.
 105. The method of claim 101, wherein the topical formulationfurther comprising a supplement for LGR6+ stem cells, wherein thesupplement is selected from the group consisting of human serum albumin,bovine serum albumin, egg albumin, plant hydrolysate, beta-cyclodextrin,glutamine, phospholipids, fibronectin, hyaluronate, plant hydrolysate,L-alanyl-L-glutamine, gelatin, vitamin E, recombinant albumin,hyaluronic acid, epidermal growth factor, fibroblast growth factor,recombinant gelatin, Tocopheryl Nicotinate, Coenzyme Q10, andubiquinone.
 106. The method of claim 101, wherein melasma is epidermalmelasma.
 107. The method of claim 101, wherein the protocol comprisesdelivery of the first defensin to skin and/or skin-associated tissuesusing a device selected from the group consisting of a laser, acousticpressure wave device, ultrasound device, a microneedle, and a hypodermicneedle.